This Phase I STTR proposal seeks funding to transfer technology from the Foundation for Applied Molecular Evolution (nonprofit) to GenoMechanix (for-profit) relating to a strategy for doing "sequencing by synthesis". This strategy involves the incorporation, in a template-directed polymerization step, of a nucleoside triphosphate having its 3'-hydroxyl group transiently blocked by an amino group, and carrying a fluorescent tag of distinctive color. The amine blocking group is small, removable under aqueous conditions, and likely to be accepted by natural DNA polymerases, especially if their performance is enhanced via directed evolution. The Phase 1 objective is to identify a procedure for optimally removing the 3'-OH blocking group under conditions where the duplex between the primer and the template remains, and the DNA is essentially under graded. To this end, reductive, oxidative, and beta-elimination reagents will be examined. The Phase 1 milestone will be passed if the removal meets the specifications (>99% complete in < 15 minutes, aqueous conditions, < 0.5 %DNA degradation). The Phase 2 project will then develop a platform for sequencing by synthesis. A working sequencing-by-synthesis system that permits parallel (for example, a 100 x 100 array) sequencing would be extremely useful, provided that the reads are >25 nucleotides in length). This would represent a major new project direction for GenoMechanix, but within its mission of providing reagents and tools for and from genomic sciences. They would also help meet the goal of the National Institutes of Health, including the goal of reducing the cost of sequencing whole genomes by one or more orders of magnitude, enhancing biomedical research at greatly reduced cost.